Application of wheat salt-sensitive gene ProDH in breeding salt-tolerant wheat

Abstract

This invention belongs to the field of plant gene editing technology and relates to the application of the wheat salt-sensitive gene ProDH in the breeding of salt-tolerant wheat. The nucleotide sequence of the aforementioned wheat salt-sensitive gene ProDH is any one of the following sequences a1 and a2: a1 is the sequence of SEQ ID No. 1-3 in the sequence listing; a2 is a nucleotide sequence with 75% or higher identity to any one of the nucleic acid sequences in SEQ ID No. 1, SEQ ID No. 2, and SEQ ID No. 3. The beneficial effects of this invention are: This invention is the first to obtain a homozygous prodh mutant without the selection marker gene by mutating the wheat ProDH gene using gene editing methods. Comparative analysis has shown that the salt tolerance of the prodh mutant wheat is significantly improved.

Description

Technical Field

[0001] This invention belongs to the field of plant gene editing technology and relates to the application of the wheat salt-sensitive gene ProDH in the breeding of salt-tolerant wheat. Background Technology

[0002] Soil salinization severely affects crop growth and yield. With the acceleration of industrialization, rising temperatures, drought, and other factors, the area of ​​salinized land is showing a further increasing trend, posing a significant threat to food security and sustainable agricultural development.

[0003] Wheat, as one of the world's three major food crops, has significant implications for human life. Cloning wheat salt tolerance negative regulatory genes, elucidating their salt tolerance mechanisms, and creating salt-tolerant wheat mutants through gene editing technology are fundamental to breeding new salt-tolerant wheat varieties, effectively utilizing saline-alkali land, and increasing grain yields. This is of great importance to ensuring food security and sustainable agricultural development.

[0004] ProDH encodes proline dehydrogenase, a key enzyme in proline catabolism; its loss of function leads to increased proline accumulation in plants. Proline plays multiple roles in plant stress responses, not only as an osmotic regulator involved in maintaining cellular water balance but also by stabilizing protein structure, scavenging reactive oxygen species, and increasing cell membrane stability to alleviate salt stress damage (Hayat et al., 2012). Currently, research on the function of the wheat ProDH gene in wheat salt tolerance is limited, and no studies have reported on creating new proDH-tolerant wheat germplasm through gene editing. Summary of the Invention

[0005] The purpose of this invention is to provide an application of the wheat salt-sensitive gene ProDH in the breeding of salt-tolerant wheat. This invention provides a wheat salt-sensitive gene ProDH, and gene editing mutation of this gene in wheat significantly improves its salt tolerance. Simultaneously, knocking out this gene in wheat promotes tillering. Therefore, knocking out ProDH can enhance wheat salt tolerance by increasing the number of tillers without affecting wheat growth and development, and has significant application potential.

[0006] The technical solution of this invention is as follows:

[0007] First, bioinformatics analysis was used to determine the coding sequence of ProDH in Jimai 22. Then, two editing target sites that simultaneously target homologous genes A, B, and D were designed in the coding region to construct a wheat ProDH gene editing vector. Finally, wheat mutants with ProDH gene knockout were obtained by infecting wheat callus tissue with Agrobacterium. Sequencing confirmed homozygous mutants. Homozygous mutants without selection markers were obtained through progeny isolation. Subsequently, salt tolerance and agronomic phenotype identification of the mutants were performed.

[0008] The wheat salt-sensitive gene provided by this invention is named ProDH, and its nucleotide sequence includes one of the following sequence features (a1) and (a2): (a1) The sequences of SEQ ID No. 1-3 in the sequence listing. SEQ ID No. 1 is the cDNA sequence of the ProDH1A (gene ID: TraesCS1A03G0552200) gene; SEQ ID No. 2 is the cDNA sequence of the ProDH1B (gene ID: TraesCS1B03G0644100) gene; SEQ ID No. 3 is the cDNA sequence of the ProDH1D (gene ID: TraesCS1D02G212400) gene.

[0009] (a2) Nucleotide sequences that are 75% or more identical to the nucleic acid sequences of SEQ ID No.1, SEQ ID No.2, and SEQ ID No.3, respectively.

[0010] The present invention also provides the protein sequence of the wheat salt-sensitive gene ProDH, which encodes a protein having one of the following sequence characteristics (b1) and (b2):

[0011] (b1) Sequences of SEQ ID No. 4-6 in the sequence listing. SEQ ID No. 4 corresponds to the ProDH1A protein sequence; SEQ ID No. 5 corresponds to the ProDH1B protein sequence; SEQ ID No. 6 corresponds to the ProDH1D protein sequence.

[0012] (b2) Proteins that have the same function by substituting and / or deleting and / or adding one or more amino acid residues of the sequences SEQ ID No. 4, SEQ ID No. 5, and SEQ ID No. 6 in the sequence listing.

[0013] The present invention also provides a gene editing vector pYLCRISPR / Cas9Pubi-B-ProDH containing the above-mentioned wheat ProDH gene.

[0014] The present invention relates to the application of the gene ProDH in the cultivation of salt-tolerant plants, wherein the plant is wheat.

[0015] Any vector that can delete the function of a foreign gene can be used, but the preferred gene editing vector of this invention is pYLCRISPR / Cas9Pubi-B-ProDH.

[0016] The gene described in this invention can be used to breed new salt-tolerant wheat varieties.

[0017] The beneficial effects of this invention: This invention is the first to obtain a homozygous prodh mutant without selection marker genes by mutating the wheat ProDH gene through gene editing. Comparative analysis has shown that the salt tolerance of the prodh mutant wheat is significantly improved. Attached Figure Description

[0018] Figure 1 Expression pattern analysis of ProDH

[0019] A: Expression pattern of ProDH gene under NaCl treatment;

[0020] B: Expression pattern of the ProDH gene under ABA treatment;

[0021] Figure 2 Agronomic traits analysis of prodh mutant wheat

[0022] A: Molecular identification of gene editing;

[0023] B: growth and development phenotype of prodh mutant; WT: wild type; prodh: mutant;

[0024] Figure 3 Salt tolerance phenotype analysis of prodh mutant wheat

[0025] A: Salt tolerance phenotypes of wheat during germination; B: Statistical analysis of primary root length of wheat (Figure A); C: Salt tolerance phenotypes of different wheat varieties during seedling stage; D: Statistical analysis of aboveground and underground fresh weight and aboveground wilting area of ​​wheat (Figure C);

[0026] Figure 4 Determination of salt tolerance physiological indicators in prodh mutant wheat. Detailed Implementation

[0027] Example 1: Expression analysis of the ProDH gene

[0028] 1.1 Material Handling

[0029] Salt-tolerant wheat seeds germinated normally and were cultured in Hangload medium for another week. For the treatment groups, 200 mM NaCl or 100 µM ABA was added to the liquid culture medium. Wheat seedling leaves were harvested at 0, 0.5, 3, 6, and 24 hours after treatment and immediately placed in liquid nitrogen for storage.

[0030] 1.2 Wheat Total RNA Extraction

[0031] The Trizol extraction kit manufactured by Invitrogen was used (the specific operating procedures were performed according to the instructions provided with the kit).

[0032] 1.3 Synthesis of First-Strand cDNA

[0033] Using PrimeScript TM Use the RT-PCR kit according to the instructions.

[0034] 1.4 qRT-PCR (real-time PCR) reaction

[0035] (1) Using cDNA from wheat roots under control and treatment conditions as templates, the CDS sequence of the target gene was submitted to the Wheatomics website for sequence alignment, thereby obtaining the CDS sequences of the three homologous genes on the chromosome. Primers for real-time PCR were designed for the three homologous genes A, B, and D. The primer sequences are as follows:

[0036] ProDH-qRT-F: 5′-CAAGATCACGGCGCTGT-3′

[0037] ProDH-qRT-R: 5′-TAGTCGATGGACGGCTGC-3′

[0038] ActinF:5′-GTTCCAATCTATGAGGGATACACGC-3′

[0039] ActinR:5′-GAACCTCCACTGAGAACAACATTACC-3′

[0040] (2) The reaction system is:

[0041] Table 1 qPCR amplification system 2 ×SYBR qPCR Master Mix 5 ProDH-qRT-F (2 μM) 1 ProDH-qRT-R (2 μM) 1 cDNA 1 <![CDATA[ddH2O]]> 2

[0042] (3) The reaction program was 94 °C for 30 s; 40 cycles of 94 °C for 15 s, 55 °C for 15 s, and 72 °C for 25 s. The qRT-PCR results are shown in (…). Figure 1 ).

[0043] Example 2: Agronomic trait analysis of prodh gene-edited mutants

[0044] 2.1 Construction and genetic transformation of ProDH gene editing vector

[0045] Based on the ProDH gene CDS sequence, two gRNA target sequences targeting three partially homologous ProDH genes were designed. Homologous recombination was used to construct the ProDH CRISPR / Cas gene knockout vector pYLC-RISPR / Cas9Pubi-B-ProDH. The vector was transformed into Agrobacterium GV3101 (psoup-p9), and then transformed into the recipient Jimai 22 callus tissue using Agrobacterium-mediated transformation. Transformed plants were obtained through differentiation and regeneration, and selected from the transformed plants.

[0046] 2.2 Molecular identification and agronomic phenotypic analysis of prodh mutant wheat

[0047] (1) Molecular identification of Prodh mutant wheat

[0048] PCR amplification of the ProDH gene sequence and sequencing identification of target site editing in gene-edited lines showed that different gene-edited lines exhibited target sequence editing, with the editing type being the deletion of multiple base sequences. Figure 2 A).

[0049] (2) Phenotypic identification of agronomic traits in prodh mutant wheat

[0050] Different wheat seeds were field-cultured to maturity, during which tiller number, flowering time, plant height, and ear type were investigated. The results showed that knocking out ProDH promoted tillering (…). Figure 2 B), heading stage, plant type, ear type ( Figure 2 B) No significant difference.

[0051] Example 3: Identification of salt tolerance phenotype in prodh mutants

[0052] After seed sterilization with 1.5% NaClO, seeds were cultured for 3 days in both normal and Hoagland medium containing 150 mM NaCl. Germination rates were recorded and root morphology differences were observed. Results showed that under normal conditions (CK), there was no significant difference in germination rates between the wild-type and the two gene-edited lines prodh 1-7 and prodh 2-4. However, under 150 mM NaCl treatment, the germination rates of the gene-edited lines were significantly higher than those of the wild-type, ranging from 11.5% to 14.4%. Figure 3 (A, B). This indicates that the ProDH mutation enhances the germination ability of wheat under salt stress.

[0053] Wild-type and gene-edited lines were cultured to the two-leaf-one-heart stage and then treated with 200 mM NaCl for 6 days. Figure 3 As shown in C, compared with the gene-edited lines, the wild-type strains exhibited more severe symptoms such as yellowing and wilting of leaves, significantly reduced fresh weight of both above-ground and below-ground parts, and a larger area of ​​wilting leaves. Figure 3 C, D).

[0054] Example 4: Determination of Salt Tolerance Physiological Indicators in prodh Mutants

[0055] 4.1 Material Treatment: Plump wheat seeds of varieties JM22, prodh 1-7, and prodh 2-4 with uniform size were selected. After disinfection with 1.5% NaClO for 20 min, the seeds were rinsed 3-5 times with sterile distilled water. The treated seeds were then soaked in distilled water containing 0.100 mM NaCl and 100 mM NaCl, respectively, for 24 h, and then germinated in a constant temperature incubator. When the plumules elongated to approximately 4 cm, seedlings with uniform growth were selected and transferred to 96-well black boxes, where they were cultured in 1 / 2 Hoagland nutrient solution containing 0 and 100 mM NaCl. Two weeks after treatment, plant phenotypes were observed, and plant height, root length, biomass, and other indicators were measured. Relevant physiological indicators were also analyzed.

[0056] 4.2 The activities of antioxidant enzymes such as SOD, POD, CAT and APX, as well as the proline assay, were performed according to the kit instructions.

[0057] 4.3 MDA content determination

[0058] Weigh 0.1 g of wheat leaves, grind them using a high-throughput tissue homogenizer, add 1 mL of 10% TCA, homogenize in an ice bath, and pour into a 10 mL centrifuge tube. Centrifuge at 4 °C and 4000 rpm for 10 min, transfer the supernatant to a new 7 mL centrifuge tube, add an equal volume of 0.67% TBA, mix, and boil in 100 °C water for 15 min. After cooling naturally to room temperature, transfer the supernatant to a new 1.5 mL centrifuge tube, centrifuge at 12000 rpm at room temperature, and collect the supernatant. Measure the absorbance of the supernatant at 450 nm, 532 nm, and 600 nm using a spectrophotometer. The malondialdehyde content (μmol / L) is calculated using the formula: 6.45 × (OD532 - OD600) - 0.56 × OD450.

[0059] 4.4 Determination of soluble sugar content

[0060] The sample preparation was the same as that for MDA content determination, but the absorbance of the supernatant was measured at a wavelength of 450 nm. Therefore, the soluble sugar content (μmol / g) = 11.71 × OD450 (μmol / g) = 11.71 × OD450.

[0061] The measurement results are as follows Figure 4As shown, under salt stress, the activities of SOD, POD, CAT, and APX in the gene-edited lines were significantly higher than those in the wild type, while the accumulation of proline and soluble sugars was also significantly higher. This indicates that the prodh mutant lines can activate a more effective antioxidant defense system and synthesize osmotic protective substances to improve wheat salt tolerance under salt stress.

Claims

1. The wheat salt-sensitive gene ProDH, characterized by, The nucleotide sequence of the wheat salt-sensitive gene ProDH is any one of the following sequences a1 and a2: Sequences of SEQ ID No. 1-3 in the a1 sequence list; SEQ ID No. 1: cDNA sequence of the ProDH1A gene; ProDH1A gene ID: TraesCS1A03G0552200; SEQ ID No. 2: cDNA sequence of the ProDH1B gene; Gene ID of ProDH1B: TraesCS1B03G0644100; SEQ ID No. 3: cDNA sequence of the ProDH1D gene; Gene ID of ProDH1D: TraesCS1D02G212400. a2 is a nucleotide sequence that is 75% or more identical to any one of the nucleic acid sequences in SEQ ID No. 1, SEQ ID No. 2, and SEQ ID No.

3.

2. The wheat salt-sensitive gene ProDH as described in claim 1, characterized in that, The protein sequence of the wheat salt-sensitive gene ProDH encodes a protein having one of the following sequence characteristics, b1 and b2: b1 is the sequence of SEQ ID No. 4-6 in the sequence list; SEQ ID No. 4 corresponds to the ProDH1A protein sequence; SEQ ID No. 5 corresponds to the ProDH1B protein sequence; SEQ ID No. 6 corresponds to the ProDH1D protein sequence; b2 is a protein that has undergone substitution and / or deletion and / or addition of one or more amino acid residues in any of the sequences in the sequence listing SEQ ID No. 4, SEQ ID No. 5, and SEQ ID No. 6 and has the same function.

3. The wheat salt-sensitive gene ProDH as described in claim 1, characterized in that, The gene editing vector for the wheat salt-sensitive gene ProDH is pYLCRISPR / Cas9Pubi-B-ProDH.

4. The application of the wheat salt-sensitive gene ProDH as described in claim 1 in the cultivation of salt-tolerant plants.

5. The application as described in claim 4, characterized in that, The plant in question is wheat.

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